Enhanced expression of transgenes

ABSTRACT

The present invention provides for improved methods of gene transfer, both in vitro and in vivo. By treating neoplastic cells with a DNA-damaging agent prior to transduction with a transgene, the expression of the transgene is improved up to about 3-fold over the expression seen in the absence of the DNA-damaging agent treatment. This effect is not dependent on the tumor cell type, the method of DNA transduction or type of DNA-damaging agent. The effect is most dramatic when the transduction is performed about 1-3 days following treatment with the DNA-damaging agent.

This is a continuation of co-pending application Serial No.PCT/US97/05325, international filing date Apr. 1, 1997, and claimspriority to U.S. provisional application 60/015,790, filed Apr. 17,1996.

The government may own rights in the present invention pursuant tofunding of research under NIH Grant No. CA66037-01.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates to the field of gene expression,generally, and more specifically it adduces the expression recombinanttransgenes in host cells. The invention may be exploited in theproduction of recombinant proteins in vitro or in gene therapy in vivo.

B. Related Art

The ability to express foreign genes in host cells has become a pivotaltool in molecular biology. For example, expressing proteins in hostcells in vitro can lead to the large scale production of the protein foruse in research or therapy. Examples of proteins that could be used inthis manner are hormones, such as insulin, or cytokines, such as theinterleukins. Scientists are constantly seeldng ways to maximizeexpression of tansgenes when they are imported into host cells.

Another important technology affected by foreign gene expression is genetherapy. Attaining high level expression in specific target cells is akey aspect of gene therapy, too. Numerous parameters have been varied inan effort to achieve higher levels of expression including the mode ofgene trasduction, the vector, the promoter, as well as the dose androutes of administration.

Recently Son & Huang (1994) reported that exposure of CDDP-resistantovarian carcinoma cells to CDDP prior to liposome-mediated gene transferresulted in enhanced gene transduction. This study utilized an ovariancancer cell line, 2008, that rapidly acquire CDDP-resistance followingexposure. The cells in this study were exposed to CDDP for four to sixweeks prior to gene transfer (in vitro) or exposed once, one week priorto gene transfer (in vivo). According to the authors, their data onlyindicate that CCDP-resistant cells show improved gene transduction.Thus, from this study, it is unclear whether CDDP-sensitive cells wouldprovide the same results. It also is unclear whether the effect was tiedto liposomal transfection methods, or could be more broadly applied.

A similar phenomenon has been observed in primary human foreskinfibroblasts infected with adeno-associated virus following briefexposure to high concentrations of CDDP or other DNA-damaging agents(Alexander et al., 1994; Russell et al., 1995). In this system, thesenon-malignant cells were rendered more susceptible to transduction bysublethal, but relatively high levels of DNA damaging agents. Thetreatments were conducted for 16 to 20 hours, followed immediately bytransduction. The cause of the increased expression was not correlatedwith other transduction methodologies. Moreover, it was unclear from thedata whether the treatment rendered the cells more competent for AAVtransduction, whether increased uptake of AAV was induced or whetherincreased expression of AAV occurred following internalization.

Thus, it is clear that the prior art does not provide a clear picture,with respect to the effect of DNA-damaging agents, of their effects onthe transduction and expression of transgenes in various host cells.There remains a need for a better understanding of these phenomena andfor increasing the expression of transgenes in transduced cells.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide improvedmethods for expression of transgenes in host cells. This applies both tothe in vitro and in vivo applications and includes a variety ofdifferent gene delivery systems, DNA-damaging agents, transgenes andtarget cells. More specifically, the invention provides for theenhancement of gene expression by providing a DNA damaging agent,preferably to a dividing cell, prior to administration of a vectorcontaining a gene or genes of interest

In one embodiment, the invention provides a method for enhancing theexpression of a transgene comprising (a) contacting a target cell with aDNA-damaging agent; (b) removing the DNA-damaging agent from the targetcell; and (c) transferring the transgene into the target cell betweenabout 1-3 days after removing the DNA-damaging agent. More preferably,the tranfser of the gene occurs at about 2 days after removal. Thetarget cell preferably is a dividing cell, and more preferably is atumor cell.

The cell may be drug sensitive or drug insensitive.

The DNA-damaging agent is selected from the group consisting ofcisplatin, carboplatin, VP16, teniposide, daunorubicin, doxorubicin,dactinomycin, mitomycin, plicamycin, bleomycin, procarbazine,nitrosourea, cyclophosphamide, bisulfan, melphalan, chlorambucil,ifosfamide, merchlorehtamine, and ionizing radiation.

Transfer of the transgene is accomplished by a technique selected fromthe group consisting of liposome-mediated transfection,receptor-mediated internalization and viral infection.

The transgene may be a tumor suppressor, such as p53. The transgene maybe under the transcriptional control of a promoter, for example, the CMVIE promoter. Further, the trangene may have, in operable relationthereto, a polyadenylation signal, for example, the SV40 polyadenylationsignal The transgene may be carried in an adenoviral vector.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A—Time Course Analysis of CDDP Treatment. Time course ofCDDP-induced enhancement of β-gal gene expression in H1299 (open bar)and H460 (hatched bar) cells in vitro. Maximal enhancement of β-gal geneexpression was observed 2 days after exposure to CDDP (*=p<0.001 byANOVA and Student's t test). Daily infection of untreated H1299 and H460cells indicated that the transduction efficiency remained unchanged overthe study period; mean±SD (day 0; immediately after removal of CDDP)(n=6; results of 2 experiments with triplicate samples). Cells werestained for β-gal expression 24 hrs after infection.

FIG. 1B—Dose Response Analysis of CDDP Treatment. Dose response curve ofCDDP-induced enhancement of β-gal gene expression in H1299 (open bar)and H460 (hatched bar) cells. Maximal enhancement of β-gal geneexpression was observed in cells exposed to 0.062 μg/ml×24 hrs(*=p<0.001 by ANOVA and Student's t test); mean±SD (n=6; results of twoexperiments with triplicate samples). Cells were stained for β-galexpression, 24 hrs after infection.

FIG. 2A—Enhancment of β-gal Gene Expression in CDDP-Treated Cells inRelation to Increasing MOI's. The β-gal activity of CDDP-treated H1299cells was quantitated by the ONPG assay, 24 hrs after incubation withAd/CMV/β-gal. Open circles represent enhancement index; filled circlesrepresent β-gal activity; mean±(n=4; results of two experiments withduplicate samples).

FIG. 2B—Enhancement of β-gal Gene Expression in CDDP-Treated Cells inRelation to Increasing MOI's. The β-gal activity of CDDP-treated H460cells was quantitated by the ONPG assay, 24 hrs after incubation withAd/CMV/β-gal. Open circles represent enhancement index; filled circlesrepresent β-gal activity; mean±(n=4; results of two experiments withduplicate samples).

FIG. 3—Enhancement of β-gal Gene Expression Following Exposure toDNA-Damaging Agents. Enhancement of β-gal expression was observedfollowing treatment with DNA-damaging agents (CDDP, Etoposid (VP16) orionizing radiation (XRT)), but not to other anti-neoplastic agents(Methotrexate (MIX), Vincristine (VIN), 5-Fluorouracil (5FU) orTransplatinum (TRANS)). Only data of cells treated with drugs (hatchedbar) at the concentration of 0.062 μg/ml or irradiated with 8 Greys areshown here along with data of non-treated cells (open bar); mean±SD(n=6; results of two experiments with triplicate samples). Cells werestained for β-gal expression, 24 hrs after infection (*=p<0.001 by ANOVAand students t test).

FIG. 4A—Effect of CDDP on Reporter Gene Expression Using DifferentVectors. Ad/CMV/β-gal (open bar), Ad/PLL/DNA complex (hatched bar) orlipofectamine (solid bar) carrying a β-gal expressing plasmid wereincubated with CDDP-treated (0.062 μg/ml for 24 hrs) H1299 cells 48 hrsafter CDDP removal; mean±SD (n=6; results of two experiments, withtriplicate samples) (*=p<0.001 by ANOVA and Student's t test). Cellswere stained for β-gal expression, 24 hrs after vector administration.

FIG. 4B—Effect of CDDP on Reporter Gene Expression Using Different CellTypes. Ad/CMV/β-gal was used to infect malignant and normal cells thatwere CDDP-treated (hatched bar) or untreated (open bar) withAd/CMV/β-gal. Infection was performed on day 2 following exposure toCDDP (0.062 μg/ml for 24 hrs) or mock exposure with PBS. Only data ofcells treated with 0.062 μg/ml CDDP are shown. MOI=1 (H1299; SiHa);MOI=5 (H460, NHBE); MOI=10 (H358, H226br, A549). Mean±SD (n=6; resultsof two experiments, with triplicate samples). Cells were stained forβ-gal expression, 24 hrs after infection.

FIG. 5—In Vivo Enhancement of β-gal Expression by SequentialAdministration of CDDP and Intratumoral Injection of Ad/CMV/β-gal. Invivo enhancement of β-gal expression by sequential administration ofCDDP (5 μg/g body weight; intraperitoneal injection) and intratumoralinjection of Ad/CMV/β-gal (5×10⁸ viral particles) on the same day (day0), 2, 4 or 6 days after CDDP administration; mean±(n=4 animals)(*=p<0.001 by ANOVA and Student's t test). Open bar=CDDP treatedanimals; hatched bar=PBS treated animals. β-gal analysis was done 24 hrsafter vector administration.

FIG. 6—Enhancement of tumor killing effect by the combination of CDDPand Ad/CMV/p53 in vitro. Cells were exposed with CDDP (0.062 μg/ml) for24 hrs, then transfected with either Ad/CMV/p53 or dl312 (MOI=5) 2 dayslater. Unexposed cells served as controls; *=p<0.001 by Student's ttest, n=5.

FIG. 7A—Superior in vivo tumor suppression of sequential intraperitonealCDDP and intratumoral Ad/CMV/p53 administrations. A divided dose regimen(1.5×10¹⁰ viral particles in three equal doses injected on alternatedays) for the administration of CDDP (solid arrow) and Ad/CMV/p53 (openarrow), or a single dose regimen (1.5×10¹⁰ viral particles in a singledose) with or without CDDP is shown (p<0.001 by Student's t test).

FIG. 7B—Superior in vivo tumor suppression of sequential intraperitonealCDDP and intratumoral Ad/CMV/p53 administrations. A single dose regimenwith CDDP given either before (CDDP on day 0, Ad/CMV/p53 on day 3),concurrent with (CDDP+Ad/CMV/p53 on day 0) or after (Ad/CMV/p53 on day0, CDDP on day 3) is shown (p<0.001 by ANOVA and Student's t test; n=5per group).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relies on the observation that treatment ofneoplastic cells with DNA-damaging agents, prior to transduction with atransgene, results in the enhanced expression of the transgene. Thiseffect is not observed when the cell is not neoplastic, i.e., when thecell exhibits normal growth control. Similarly, the effect is notobserved when non-DNA-damaging anti-neoplastic agents are used. However,the effect does not appear to be limited to particular transgenes nor isit limited to particular transduction methodology, particular neoplasticcells or particular DNA-damaging agents. The invention is described indetail in the following sections.

A. Sequential Administration of a DNA-Damaging Agent and a Transgene

In accordance with the present invention, the methods provided for theenhancement of transgene expression are both time and order dependent.Each of these phenomena are discussed below and illustrated in theexamples. These data should not be construed as indicating that timeframes or orders outside the preferred embodiments of the presentinvention are inoperable; to the cony, data show that simultaneousadministration of the p53 tumor suppressor gene in an adenoviral vectorand cisplatin results in improved killing of tumor cells, when comparedto treatment with the p53 adenoviral vector alone. It is the presentinventors' observation, however, that using a particular order and usingparticular timing, transgene expression may be enhanced over thatobserved with other protocols.

As the data of the instant examples show, the prior treatment of hostcells with a DNA-damaging, followed by provision of a transgene, resultsin improved expression of the transgene when compared to simultaneous orsubsequent treatment with a DNA-damaging agent, or no DNA-damaging agentat all. In addition, there are particular time frames in which thiseffect is maximized. Preferably, the in vitro administration of theDNA-damaging agent precedes the transduction of the host cell by about1-3 days (about 24-72 hrs) and, more preferably, about 2 days (about 48hrs) after removal of the agent. In vivo, the time frame may be delayedsomewhat, depending on the type and route of administration. Thus, it issuspected that a systenic administration of a chemotherapeutic shouldprecede provision of the transgene by 2-4 days (about 48-96 hrs) and,more preferably, about 3 days (72 hrs).

Some minor variation in the optimal treatment times is expecteddepending on the tumor cell type, the particular transgene, the route ofadministration, the DNA-damaging agent or the delivery vector. To theextent that the skilled artisan desires, the optimal timing may beascertained by performing time course experiments like those set outbelow in the examples to determine what times will provide the bestexpression in a given system. It is expected, however, that most willfall within the time frames set forth above.

B. DNA Damaging Agents

As stated above, the present invention relies on the ability ofDNA-damaging agents to facilitate enhanced transgene expression in hostcells. For the purposes of this application, DNA-damaging agents aredefined as those agents that cause structural changes in DNA strandsexisting in the target cell at the time of. The following agents areprovided as exemplary of DNA-damaging agents, as defined herein:cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan,nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicomycin, mitomycin, etoposide (VP16), and ionizing radiation. Otheragents may be included as long as the adhere to the definition ofDNA-damaging agents, provided above.

The following agents are not classified as DNA-damaging agents accordingto the present invention: transplatinum, 5-fluorouracil, vincristin,vinblastin and methotrexate.

Cisplatin (cis-dichlorodinminocisplatin; Mw=300.05) is a heavy metalcompound used as a cancer chemotherapeutic. It has been applied to thetreatment of testicular, ovarian, bladder and head/neck cancers. CDDPcauses intra- and inter-strand crosslinking of DNA by forming guanineadducts and eventually DNA strand breaks (Eastman, 1990). CDDP isusually administered in 6-8 h periods with 1 liter of fluid and 25 to 50mg of mannitol. The total dose administered usually is 40-120 mg/m² pertherapy cycle, but depends on the frequency of cycles and individualtolerance. A common schedule is 20 mg/m²/day for five days.

Carboplatin has a mechanism of action and spectrum of clinical activitysimilar to CDDP. It is often employed in treating recurrent ovariancancer, including patients who have previously received cisplatin.Administration is an intravenous infusion over at least 15 min. Theusual dose is 360 mg/², given once every four weeks.

Procarbazine, a methyl hydrazine derivative, is often used clinicallyagainst Hodgkin's disease, and also has shown activity against brain andlung tumors, non-Hodgkin's lymphoma, melanoma and myeloma. This druginduces chromosomal damage and chromatid breaks and translocations. Therecommended oral daily dose for adults is 100 mg/m² for ten days incombination regimens.

Mechlorethamine is a nitrogen mustard that is used in treating Hodgkin'sand other lymphomas. Among the effects observed with various nitrogenmustard agents is the opening of imidazole rings or depurination ofbases in DNA, both of which can result in serious damage to the DNAstrand. The course of therapy consists of the injection of a total doseof 0.4 mg/kg or 10 mg/m². This total dose may be given in either two orfour daily consecutive injections, a single administration ispreferable. Direct intracavitary administration (0.2 to 0.4 mg/kg) formalignant effusions, particularly of pleural origin, is performed.

Cyclophosphamide, another nitrogen mustard, also alkylates DNA andresults in damage to the strands. This drug has been used againstHodgkin's lymphoma, Burkitt's lymphoma and lymphoblastic leukemia. Itcan be administered orally, intravenously, intramuscularly,intrapleurally and intraperitoneally. A conservative daily dose of 2-3mg/kg, orally or intravenously, is recommended for patients with moresusceptible neoplasms such as lymphomas or leukemias. A higher dosage(4-8 mg/kg daily for six days followed by an oral maintenance dose of1-5 mg/kg daily) has been used for the treatment of carcinomas and othermore resistant neoplasms.

Melphalan is a phenylalanine derivative of nitrogen mustard. It has beenused in the treatment of multiple myeloma, malignant melanoma and incarcinoma of the breast and ovary. The usual dose for multiple myelomais 6 mg daily for a period of 2 to 3 weeks.

Ifosfamide is an analog of cyclophosphamide. It is currently approvedfor use in combination with other drugs for treatment of germ celltesticular cancer, and clinical trials have shown activity againstcarcinomas of the lung and cervix, Hodgkin's and non-Hodgkin'slymphomas, and certain sarcomas. The drug usually is infusedintravenously over 30 min at a dose of 1.2 g/m² per day for five days.Patients should also receive at least 2 l of oral or intravenous fluiddaily. Treatment cycles are usually repeated every three weeks.

Chlorambucil is an alkylating agent that has been used against chroniclymphocytic leukemia and primary macroglobulinemia. The standard initialdaily dosage is 0.1 to 0.2 mg/kg, continued for at least 3 to 6 weeks.The total daily dose, usually 4 to 10 mg, is given at one time. Bisulfanis another alkylating agent that is used to treat chronic granulocyticleukemia

Nitrosourea is clinically active against lymphomas, malignant melanomas,brain neoplasms and gastrointestinal carcinomas. Chemical decompositionsyields reactive intermediates that form single-strand adducts with DNAand then, through a dehalogenation event, forms a second reactive siteand cross-links DNA.

Dactinomycin is a antibiotic isolated from Steptomyces. In addition toblocking transcription, it induces single-strand breaks in DNA, possiblythrough a free-radical intermediate or as a result of topoisomerase II.It has been used against Hodgkin's and non-Hodgkin's lymphomas. Thetypical daily dose is 10 to 15 μg/kg/day intravenously for five days.Additional courses may be given at 3- to 4-week intervals in the absenceof toxicity. Daily injections of 100 to 400 μg have been given tochildren for up to fourteen days. Weekly maintenance doses of 7.5 μg/kghave been used.

Daunorubicin is an anthracycline antibiotic that, among other actions,induces single- and double-stranded breaks. It is used against acutelymphocytic and acute granulocytic leukemias, acute non-lymphoblasticleukemias and certain lymphomas. The recommended dosage is 30 to 60mg/m² daily for three days.

Doxorubicin is a hydroxy analog of daunorubicin, with activity againstacute leukemias and malignant lymphomas; it also is active against anumber of solid tumors, including breast cancer. The recommended dose is60 to 75 mg/m², administered as a single intravenous infusion, repeatedafter 21 days.

Bleomycins are an important group of antitumor agents derived fromStreptomyces verticillus. The currently used drug is a mixture ofcopper-chelating glycopeptides that consist primarily of two closelyrelated agents, bleomycin A₂ and bleomycin B₂. They show a wide range ofactivity against a variety of tumors, including squamous carcinomas ofthe skin, head, neck and lungs, as well as against lymphomas andtesticular tumors. Administration is a bolus dose of 15 units/m², twicea day for five days.

Plicamycin is a cytotoxic antibiotic isolated from cultures ofStreptomyces tanashiensis. It has been used against advanced embryonaltumors of the testes. The recommended dosage for treatment of testiculartumors is 25 to 30 μg/kg daily or on alternate days for eight to tendoses. It usually is administered via intravenous infusion over 4 to 6hours.

Mitomycin is an antibiotic isolated from Steptomyces caespitosus. Thedrug both inhibits DNA synthesis and cross-links DNA; single-strandbreaks are induced by the removal of mitomycin. It is used in thepalliative treatment of gastric adenocarcinoma, in conjunction with 5-FUand doxorubicin. It also has produced temporary benefits in carcinomasof the cervix, colon, rectum, pancreas, breast, bladder, head and neck,and lung, and in melanomas.

Etoposide (Mw=588.58) is a semi-synthetic derivative of podophyllotoxinwith antineoplastic activity against testicular tumors and small-cellcarcinoma of the lung, usually in combination with cisplatin. It also isactive against non-Hodgkin's lymphomas, acute non-lymphocytic leukemia,carcinoma of the breast and Kaposi's sarcoma. Etoposide forms a tertiarycomplex between DNA and topoisomerase II and eventually producesprotein-linked DNA single and double strand breaks (Kaufman, 1989).Intravenous dosage for testicular tumors is 50-100 mg/m² for five days,or 100 mg/m² on alternate days for three doses. For small-cellcarcinoma, the dose is 35 mg/m² daily for four days to 50 mg/m² dailyfor five days. If given orally, the dosage should be doubled. Cycles oftherapy are usually repeated every three to four weeks.

Teniposide is a thiophene derivative of etoposide that is used in thetreatment of refractory acute lymphblastic leukemia in children.Administration is via intravenous infusion in doses that range from 50mg/m² per day for five days to 165 mg/m² per day, twice weekly.

Radiation is an important tool in tumor therapy. Radiation falls intotwo major categories—electromagnetic radiation (waves of varyingfrequency, e.g., x-rays) and subatomic particle radiation (alpha, beta(electron), neutron, proton, meson and heavy ion). Gamma emissions are aform of electromagnetic radiation emitted from radioactive isotopes ofradium, cobalt and other elements.

Radiation therapy transfers discrete energy units, called photons, totissues causing damage to both normal and malignant cells. Ionizingirradiation stimulates production of oxygen free radicals which reactwith macromolecules and induces DNA damage (Cole et al., 1980). “Early”radiation effects include damage to proliferating cells, while “late”effects involve cell death and affect many different kinds of cells.Fortunately, radiation exploits the differential effects on malignantversus non-malignant cells, namely, that rapidly proliferating cellsundergoing significant DNA synthesis suffer more severe effects from theDNA damage induced by radiation.

The dose of radiation is dependent upon tissue and tumor type. Thetreatment is usually fractionated to prevent toxicity and can be in the1-5 Grey range over a several week period. For the treatment of rectalcancer radiation, 45 Grey total dose is given (1.8 Grey dose/day, Mondaythrough Friday). For the enhancement of gene expression, a single doseof between 1 and 8 Grey is contemplated for gamma radiation

C. Transgenes and Expression Constructs

The transgenes of the present invention may encode any protein ofinterest.

Various proteins are useful for their biological activities in vitro andin vivo. For example, cytokines and hormones have already found use intreating certain diseases. For example, the interleukins (IL-1 to 11),interferon, human growth hormone, insulin, insulin-like growth factor,prolactin, placental lactogen, luteinizing hormone, follicle stimulatinghormone, chorionic gonadotropic, thyroid-stimulating hormone, glucagon,somatostatin, calcitonin, vasopressin, vasostatin, vasotocin, gastrn,amylin, growth hormone releasing factor, growth hormone releasinghormone, luteinizing hormone releasing hormone, thymidine kinase,interleukin-1-beta converting enzyme and others are suitable subjects ofthe present invention.

In addition, the use of tumor suppressor genes such as p53, C-CAM,retinoblastoma gene, herpesvirus and p16 are candidates for genetransfer. These proteins act to regulate cell growth, and theirreduction, mutation or deletion can affect the ability of cells toundergo normal cell senescence or apoptosis. Introducing transgenesencoding these proteins can reestablish normal cell growth, andexpression of high levels of these proteins can even overcome defects inother genes that cause neoplastic proliferation.

p53 is a 53 kD nuclear phosphoprotein of 375 amino acids that controlscell proliferation. Mutations to the p53 gene, and allele loss onchromosome 17p, where this gene is located, are among the most frequentalterations identified in human malignancies. The p53 protein is highlyconserved through evolution and is expressed in most normal tissues.Wild-type p53 has been shown to be involved in control of the cell cycle(Mercer, 1992), transcriptional regulation (Fields et al., 1990; Mietzet al, 1992), DNA replication (Wilcock and Lane, 1991; Bargonetti etal., 1991) and induction of apoptosis (Yonish-Rouach et al., 1991; Shawet al., 1992).

Various mutant p53 alleles are known in which a single base substitutionresults in the synthesis of proteins that have quite different growthregulatory properties and, ultimately, lead to malignancies (Hollsteinet al., 1991). In fact, the p53 gene has been found to be the mostfrequently mutated gene in common human cancers (Hollstein et al., 1991;Weinberg, 1991), and is particularly associated with those cancerslinked to cigarette smoke (Hollstein et al., 1991; Zakut-Houri et al.,1985). The overexpression of p53 in breast tumors has also beendocumented (Casey et al., 1991).

Unlike other oncogenes, however, p53 point mutations are known to occurin at least 30 distinct codons, often creating dominant alleles thatproduce shifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encodingwild-type p53 into two human breast cancer cell lines restores growthsuppression control in such cells (Casey et al., 1991). A similar effecthas also been demonstrated on transfection of wild-type, but not mutant,p53 into human lung cancer cell lines (Takahasi et al., 1992). The p53appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 does not affect the growth of cellswith endogenous p53.

Alternatively, the transgenes may encode antisense oligonucleotides thathybridize, under intracellular conditions, to a target nucleic acid. Thetarget nucleic acid may be a DNA molecule or an RNA molecule.Hybridization results in the inhibition of transcription and ortranslation of the protein encoded by the target nucleic acid The designof antisense constructs, based on the sequence of genes, will be evidentto those of skill in the art

The term “antisense nucleic acid” is intended to refer to theoligonucleotides complementary to the base sequences of a target DNA orRNA. Antisense oligonucleotides, when introduced into a target cell,specifically bind to their target nucleic acid and interfere withtranscription, RNA processing, transport, translation and/or stability.Targeting double-stranded (ds) DNA with oligos or oligonucleotides leadsto triple-helix formation; targeting RNA will lead to double-helixformation.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNA's,may be employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject. Nucleic acid sequences which comprise“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,that the larger purines will base pair with the smaller pyrimidines toform combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA. Inclusion of less common basessuch as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine andothers in hybridizing sequences does not interfere with pairing.

As used herein, the terms “complementary” or “antisense sequences” meannucleic acid sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, nucleicacid sequences of fifteen bases in length may be termed complementarywhen they have a complementary nucleotide at thirteen or fourteenpositions with only a single mismatch. Naturally, nucleic acid sequenceswhich are “completely complementary” will be nucleic acid sequenceswhich are entirely complementary throughout their entire length and haveno base mismatches.

Other sequences with lower degrees of homology also are contemplated.For example, an antisense construct which has limited regions of highhomology, but also contains a non-homologous region (e.g., a ribozyme)could be designed. These molecules, though having less than 50%homology, would bind to target sequences under appropriate conditions.

While all or part of the gene sequence may be employed in the context ofantisense construction, statistically, any sequence of 17 bases longshould occur only once in the human genome and, therefore, suffice tospecify a unique target sequence. Although shorter oligomers are easierto make and increase in vivo accessibility, numerous other factors areinvolved in determining the specificity of hybridization. Both bindingaffinity and sequence specificity of an oligonucleotide to itscomplementary target increases with increasing length. It iscontemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more base pairs will be used. One can readilydetermine whether a given antisense nucleic acid is effective attargeting of the corresponding host cell gene simply by testing theconstructs in vitro to determine whether the endogenous gene's functionis affected or whether the expression of related genes havingcomplementary sequences is affected.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines. Oligonucleotides which contain C-5 propyneanalogues of uridine and cytidine have been shown to bind RNA with highaffinity and to be potent antisense inhibitors of gene expression(Wagner et al., 1993).

As an alternative to targeted antisense delivery, targeted ribozymes maybe used. The term “ribozyme” is refers to an RNA-based enzyme capable oftargeting and cleaving particular base sequences in target DNA and RNA.Ribozymes can either be targeted directly to cells, in the form of RNAoligonucleotides incorporating ribozyme sequences, or introduced intothe cell as an expression construct encoding the desired ribozymal RNA.Ribozymes may be used and applied in much the same way as described forantisense nucleic acids. Ribozyme sequences also may be modified in muchthe same way as described for antisense nucleic acids. For example, onecould incorporate non-Watson-Crick bases, or make mixed RNA/DNAoligonucleotides, or modify the phosphodiester backbone.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA)as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA does not contain any non-coding sequencesbut, rather, contains only the coding region of the correspondingprotein. There may be times when the full or partial genomic sequence ispreferred, such as where the non-coding regions are required for optimalexpression or where non coding regions such as introns are to betargeted in an antisense strategy.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. Thus, in certainembodiments, expression includes both transcription of a gene andtranslation of a RNA into a gene product In other embodiments,expression only includes transcription of the nucleic acid, i.e.,antisense and ribozymes.

In preferred embodiments, the nucleic acid is under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the cell, or introduced syntheticmachinery, required to initiate the specific transcription of a gene.The phrase “under transcriptional control” means that the promoter is inthe correct location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can finction either co-operatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell. Generally spealing, such a promotermight include either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) inmediate earlygene promoter, the SV40 early promoter and the Rous sarcoma virus longterminal repeat can be used to obtain high-level expression oftransgenes. The use of other viral or mammalian cellular or bacterialphage promoters which are well-known in the art to achieve expression ofa transgene is contemplated as well, provided that the levels ofexpression are sufficient for a given purpose.

By employing a promoter with well-known properties, the level andpattern of expression following transfection can be optimized. Forexample, selection of a promoter which is active specifically in lungcells, such as tyrosinase (melanoma), alpha-fetoprotein and albumin(liver tumors), CC10 (lung tumor) and prostate-specific antigen(prostate tumor) will permit tissue-specific expression. Further,selection of a promoter that is regulated in response to specificphysiologic signals can permit inducible expression. For example, withhuman PAI-1 promoter, expression is inducible by tumor necrosis factor.Tables 1 and 2 list several elements/promoters which may be employed, inthe context of the present invention, to regulate the expression of atransgene. This list is not intended to be exhaustive of all thepossible elements involved in the promotion of transgene expression but,merely, to be exemplary thereof.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Below is a list of viral promoters, cellular promoters/enhancers andinducible promoters/enhancers that could be used in combination with thenucleic acid encoding a transgene in an expression construct (Table 1and Table 2). Additionally any promoter/enhancer combination (as per theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression of a transgene. Use of a T3, T7 or SP6 cytoplasmic expressionsystem is another possible embodiment Eukaryotic cells can supportcytoplasmic transcription from certain bacterial promoters if theappropriate bacterial polymerase is provided, either as part of thedelivery complex or as an additional genetic expression construct.

TABLE 1 PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light ChainT-Cell Receptor HLA DQ α and DQ β β-Interferon Interleukin-2Interleukin-2 Receptor MHC Class II 5 MHC Class II HLA-DRα β-ActinMuscle Creatine Kinase Prealbumin (Transthyretin) Elastase IMetallothionein Collagenase Albumin Gene α-Fetoprotein τ-Globin β-Globinc-fos c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM)α_(1-Antitrypsin) H2B (TH2B) Histone Mouse or Type I CollagenGlucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone HumanSerum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived Growth FactorDuchenne Muscular Dystrophy SV40 Polyoma Retrovinises Papilloma VirusHepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus GibbonApe Leukemia Virus

TABLE 2 Element Inducer MT II Phorbol Ester (TFA) Heavy metals MMTV(mouse mammary Glucocorticoids tumor virus) β-Interferon poly(rI)Xpoly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ CollagenasePhorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 PhorbolEster CITA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kBInterferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPATumor Necrosis Factor FMA Thyroid Stimulating Hormone Thyroid Hormone αGene

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Also contemplated as an element of the expression cassette is aterminator. These elements can serve to enhance message levels and tominimize read through from the cassette into other sequences.

Specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancer elements(Bittner et al., 1987).

In preferred embodiments of the invention, the expression constructcomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and to integrate into host cell genome and express viralgenes stably and efficiently have made them attractive candidates forthe transfer of foreign genes into mammalian cells (Ridgeway, 1988;Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).The first viruses used as gene vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986), adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986). These have a relatively low capacityfor foreign DNA sequences and have a restricted host spectrum.Furthermore, their oncogenic potential and cytopathic effects inpermissive cells raise safety concerns. They can accommodate only up to8 kilobases of foreign genetic material but can be readily introduced ina variety of cell lines and laboratory animals (Nicolas and Rubenstein,1988; Temin, 1986).

(i) Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene, termed Ψ, functions as a signal for packaging of the genomeinto virions. Two long terminal repeat (LTR) sequences are present atthe 5′ and 3′ ends of the viral genome. These contain strong promoterand enhancer sequences and are also required for integration in the hostcell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding atransgene is inserted into the viral genome in the place of certainviral sequences to produce a virus that is replication-defective. Inorder to produce virions, a packaging cell line containing the gag, pol,and env genes but without the LTR and Ψ components is constructed (Mannet al., 1983). When a recombinant plasmid containing a human cDNA,together with the retroviral LTR and Ψ sequences is introduced into thiscell line (by calcium phosphate precipitation for example), the Ψsequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).The media containing the recombinant retrovinuses is then collected,optionally concentrated, and used for gene tansfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells (Paskind etal., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intact Ψsequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

One limitation to the use of retrovirus vectors in vitvo is the limitedability to produce retroviral vector titers greater than 10⁶ infectiousU/mL. Titers 10- to 1,000-fold higher are necessary for many in vivoapplications.

(ii) Adenovirus

Knowledge of the genetic organization of adenovirus, a 36 kB, linear anddouble-stranded DNA virus, allows substitution of a large piece ofadenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz1992). In contrast to retrovirus, the infection of adenoviral DNA intohost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in the human.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range, and high infectivity. Both ends of the viral genomecontain 100-200 base pair (bp) inverted terminal repeats (ITR), whichare cis elements necessary for vital DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication These proteinsare involved in DNA replication, late gene expression, and host cellshut off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence which makes them preferred mRNAs for translation.

In the current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure. Use of the YAC system is an alternative approachfor the production of recombinant adenovirus.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham, et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the E3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury, et al.,1987), providing capacity for about 2 extra kB of DNA. Combined with theapproximately 5.5 kB of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1 deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available adenovirusvectors at high multiplicities of infection (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be cmcial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in themethod of the present invention This is because Adenovirus type 5 is ahuman adenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical adenoviral vector according to the presentinvention is replication defective and will not have an adenovirus E1region. Thus, it will be most convenient to introduce the transgenenucleic acid into the position from which the E1 coding sequences havebeen removed. However, the position of insertion of the coding regionwithin the adenovirus sequences is not critical to the presentinvention. The nucleic acid encoding a transcription unit also may beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed previously by Karlsson et. al. (1986) or in the E4 regionwhere a helper cell line or helper virus complements the E4 defect

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹ plaque-forming unit (PFU)/ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal, and therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Pernicaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Experiments in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injection (Herz and Gerard, 1993), andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).In addition to these protocols, the present invention also contemplatesdirect tumoral injection.

(iii) Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesvises may be employed. They offerseveral attractive features for various mammalian cells (Friedmann,1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988;Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

D. Methods of Gene Transfer

In order to effect expression of sense or antisense transgeneconstructs, the expression construct must be delivered into a cell. Thisdelivery may be accomplished in vitro, as in laboratory procedures fortransforming cells lines, or in vivo or ex vivo, as in the treatment ofcertain disease states. As described above, the one mechanism fordelivery is via viral infection where the expression consct isencapsidated in an infectious viral particle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

In one embodiment of the invention, the expression construct may simplyconsist of naked recombinant DNA or plasmids. Transfer of the constructmay be performed by any of the methods mentioned above which physicallyor chemically permeabilize the cell membrane. This is particularlyapplicable for transfer in vitro but it may be applied to in vivo use aswell. Dubensky etal. (1984) successfully injected polyomavirus DNA inthe form of CaPO₄ precipitates into liver and spleen of adult andnewborn mice demonstrating active viral replication and acute infection.Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of CaPO₄ precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga transgene may also be transferred in a similar manner in vivo andexpress the corresponding protein.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment Thismethod depends on the ability to accelerate DNA coated microprojecfilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,ie., ex vivo treatment Again, DNA encoding a transgene may be deliveredvia this method and still be incorporated by the present invention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a transgene into cells are receptor-mediated deliveryvehicles. These take advantage of the selective uptake of macromoleculesby receptor-mediated endocytosis in almost all eukaryotic cells. Becauseof the cell type-specific distribution of various receptors, thedelivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agentSeveral ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and tansferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid may be specifically deliveredinto a cell type such as lung, epithelial or tumor cells, by any numberof receptor-ligand systems with or without liposomes. For example,epidermal growth factor (EGF) may be used as the receptor for mediateddelivery of a nucleic acid encoding a transgene in many tumor cells thatexhibit upregulation of EGF receptor. Mannose can be used to target themannose receptor on liver cells. Also, antibodies to CD5 (CLL), CD22(lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly beused as targeting moieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells, invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues. Anderson et al., U.S. Pat. No.5,399,346, and incorporated herein in its entirety, disclose ex vivotherapeutic methods.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

During in vitro culture conditions, the expression construct delivers anucleic acid to the cells and the protein is expressed. Finally, thecells may be reintroduced into the original animal, or administered intoa distinct animal, in a pharmaceutically acceptable form by any of themeans described below. Thus, providing an ex vivo method of treating amammal with a pathologic condition is within the scope of the invention.

E. Recombinant Protein Production In Vitro

Another embodiment of the present invention involves the use of genetransfer to generate recombinant cells lines in vitro for the productionof recombinant proteins. The gene of interest may be transferred asdescribed above into appropriate host cells followed by culture of cellsunder the appropriate conditions. The gene for virtually any polypeptidemay be employed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3,RIN and MDCK cells. In addition, a host cell strain may be chosen thatmodulates the expression of the inserted sequences, or modifies andprocess the gene product in the manner desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,the herpes simplex virus thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase and adenine phosphoribosyltransferase genes,in tk-, hgprt- or aprt- cells, respectively. Also, anti-metaboliteresistance can be used as the basis of selection for dhfr, that confersresistance to; gpt, that confers resistance to mycophenolic acid; neo,that confers resistance to the aminoglycoside G418; and hygro, thatconfers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (ie., a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent cells.

Large scale suspension culture of mammalian cells in stirred tanks is acommon method for production of recombinant proteins. Two suspensionculture reactor designs are in wide use—the stirred reactor and theairlift reactor. The stirred design has successfully been used on an8000 liter capacity for the production of interferon (Phillips et al.,1985; Mizrahi, 1983). Cells are grown in a stainless steel tank with aheight-to-diameter ratio of 1:1 to 3:1. The culture is usually mixedwith one or more agitators, based on bladed disks or marine propellerpatterns. Agitator systems offering less shear forces than blades havebeen described. Agitation may be driven either directly or indirectly bymagnetically coupled drives. Indirect drives reduce the risk ofmicrobial contamination through seals on stirrer shafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gases and generatesrelatively low shear forces.

F. In Vivo Gene Therapy Applications

In another embodiment of the present invention, methods for improvedgene therapy are provided. The present invention contemplates the use ofgene therapeutic vectors, in conjunction with DNA-damaging agenttreatment, to provide for high level expression of transgenes in vivo.This will be accomplished by treating an individual with an effectiveamount of a DNA damaging agent, followed by administration of atherapeutic gene. As stated above, the timing of the administration isimportant for achieving maximal enhancement of expression. The 1-3 daytime period between administration of the DNA-damaging agent andadministration of the transgene for in vitro may be delayed somewhat forin vivo applications, especially where the DNA-damaging agent isadministered systematically. However, where the DNA-damaging agent isgiven locally to the site of administration of the transgene, the 1-3window should be appropriate.

Administration of the DNA-damaging agent to patient will followwell-established protocols. For example, the administration of cisplatinwould occur via intravenous infusion over about 8 hours with adequateprior hydration to minimize nephrotoxicity. The particular dose isdependent upon tumor type and size and the overall condition of thepatient. The dose ranges from 60 mg/m² to 100 mg/m². Two to three daysfollowing cisplatin treatment, the vector system would be administered,for example, via intratumoral injection. It is anticipated that thetreatment cycles would be repeated every four weeks as necessary.

Where clinical application of an expression construct comprising anucleic acid encoding a transgene is contemplated, it will be necessaryto prepare the complex as a pharmaceutical composition appropriate forthe intended application. Generally this will entail preparing apharmaceutical composition that is essentially free of pyrogens, as wellas any other impurities that could be harmful to humans or animals. Onealso will generally desire to employ appropriate salts and buffers torender the complex stable and allow for complex uptake by target cells.

Aqueous compositions of the present invention comprise an effectiveamount of the expression construct and nucleic acid, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions can also be refered to as inocula The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The expression constructs and delivery vehicles of the present inventionmay include classic pharmaceutical preparations. Administration oftherapeutic compositions according to the present invention will be viaany common route so long as the target tissue is available via thatroute. This includes oral, nasal, buccal, rectal, vaginal or topical.Topical administration would be particularly advantageous for treatmentof skin cancers, to prevent chemotherapy-induced alopecia or otherdermal hyperproliferative disorder. Alternatively, administration willbe by orthotopic, intradermal subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositionsthat include physiologically acceptable carriers, buffers or otherexcipients.

The therapeutic compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsied. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to well known parameters.

Additional formulations are suitable for oral administration Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

An effective amount of the therapeutic agent is determined based on theintended goal, for example, in the case of tumor therapy: (i) inhibitionof tumor cell proliferation or (ii) elimination of tumor cells. The term“unit dose” refers to physically discrete units suitable for use in asubject, each unit containing a predetermined-quantity of thetherapeutic composition calculated to produce the desired responses,discussed above, in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theprotection desired. Precise amounts of the therapeutic composition alsodepend on the judgment of the practitioner and are peculiar to eachindividual.

G. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1: ENHANCEMENT OF GENE EXPRESSION IN HUMAN CARCINOMA CELLS BYDNA-DAMAGING AGENTS FOLLOWING GENE TRANSFER Materials and Methods

Cells and Culture Conditions

The human non-small cell lung carcinoma cell lines H1299 and H460 weregrown in RPMI-1640 medium supplemented with 5% heat-inactivated fetalcalf serum (FCS), 10 mM glutamine, 100 units/ml of penicillin, 100 μg/mlof streptomycin and 0.25 μg/ml of amphotericin B. Human non-small celllung carcinoma cell lines H358, H226br, and the cervical cancer cellline SiHa were maintained in complete RPMI-1640 medium supplemented with10% ECS. The cell line A549 (human, non-small cell lung carcinoma) wasgrown in Ham's F12 nutrient mixture solution (Gibco BRL, Gaithersburg,Md.) similarly supplemented with antibiotics, glutamine and 10% FCS.Primary normal human bronchial epithelial (NHBE) cells (CloneticsCorporation, San Diego, Calif.), were grown in serum-free optimizedgrowth medium and subcultured under conditions suggested by themanufacturer.

In Vitro Characterization of the Effects of CDDP Exposure on β-galExpression

Gene Delivery Protocol: In vitro gene delivery was performed byincubating cells in six-well plates (Falcon Plastics, Lincoln Park,N.J.) for two hours with Adv/CMV/β-gal in appropriate mediumsupplemented with 2% FCS. The multiplicity of infection (MOI: number ofviral particles per target cell) was based on cell counts of untreatedwells. Fresh, complete medium with the appropriate concentration of FCSwas then added to the wells at the end of the infection period and cellswere then incubated for 20 hours at 37° C., 5% CO₂. After washing thecells with ice cold PBS, the cells were fixed with ice-cold 1.25%glutaraldehyde and stained with X-gal(5-bromo-4-chloro-3-indolyl-β-d-galactoside; Gibco BRL) as previouslydescribed (MacGregor et al., 1987). The transduction efficiency byAdv/CMV/β-gal was determined by the percentage of positive stained cells(blue) for β-galactosidase activity (1000 cells counted per well).

Time Course and Dose Response: To determine the time course ofenhancement, H1299 and H460 cells were seeded in 6-well plates (2×10⁵cells/2 mls/well). After an overnight incubation period (37° C., 5%CO₂), the initial medium was replaced with freshly prepared media (2 ml)containing 0.062 μg/ml CDDP (Sigma Chemicals, St. Louis, Mo.) (CDDP wasprepared fresh by dissolving in distilled water) and the cells were thenincubated for 24 hours at 37° C., with 5% CO₂. CDDP-treated cells werethen washed twice with PBS and subsequently infected with Ad/CMV/β-gal(MOI=1 for H1299 and MOI=5 for H460) on days 0, 1, 2, 3, 4, 5 or 6following CDDP removal, to determine the time point at which maximalenhancement of expression was occurring. To establish the dose of CDDPneeded to produce maximum enhancement, the cells were treated withdifferent concentrations of CDDP (serial, 1:4 dilutions, ranging from 4μg/ml to 0.0002 μg/ml, 5 ml per 60 mm dish) for 24 hours andsubsequently incubated with Ad/CMV/β-gal 48 hours after removal of CDDP.To study if exposure of malignant cells to different classes ofantineoplastic agents would lead to a similar elevation of geneexpression, H1299 cells were treated with vincristine (Eli Lilly and Co,Indianapolis, Ind.), methotrexate (Lederle Pharmaceuticals, Pearl River,N.Y.), 5-fluorouracil (SoloPak Laboratories, Elk Grove Village, Ill.),epotoside (Bristol Laboratories, Princeton, N.J.) and transplatin (theisomer of CDDP that has no anticancer activity, Sigma Chemical, St.Louis, Mo.) in the similar fashion as CDDP treatment and then infectedwith Ad/CMV/β-gal 48 hours after removal of drug-containing media. H1299cells were treated with 2, 4, 8, or 16 Grey (Gy) of ionizing irradiationand then similarly infected 72 hours after irradiation. Untreated cellsthat were similarly infected, served as controls.

Gene Transfer Vectors: The recombinant adenovirus carrying theEscherichia coli β-galactosidase gene under the control of the humancytomegalovirus enhancer/promotor (Ad/CMV/β-gal) or the replicationdefective adenovirus were propagated on 293 cells and purified andstored using techniques previously reported (Nguyen et al., 1996). Viraltiter was determined by UV spectrophotometric analysis (Nguyen et al.,1996a; 1996b). To determine if the CDDP-induced enhancement of geneexpression would be dependent on the method of gene delivery, two othergene delivery systems were used: the Ad/PLL/DNA complex (Nguyen et al.,1996a; 1996b) and cationic liposomes (Lipofectamine, Gibco BRL). CDDP(0.062 μg/ml)-treated H1299 cells were incubated with either genedelivery system carrying a plasmid that contained the β-gal gene underthe control of the CMV enhancer/promotor (CMV/β-gal) 48 hours after drugremoval. Plasmid DNA was isolated by the alkaline lysis technique andpurified by using Qiagen DNA purification kits (Qiagen, Chatsworth,Calif.). Plasmid containing liposome preparations and gene transfer wereperformed according to the manufacturer's protocol. The conjugatedAd/PLL/DNA complex was constructed and used to deliver the β-gal gene toH1299 and H460 cells as described elsewhere (Nguyen et al., 1996a;1996b).

Quantitation of β-gal Gene Expression: Adenoviral infection wasperformed 2 days after exposure to CDDP (0.062 μg/ml×24 hours), withcell extracts being obtained 24 hours later and quantitatively assayedfor the expression of the β-galactosidase gene using O-nitrophenylβ-d-galactoside (ONPG) as a substrate (MacGregor et al., 1987). Themagnitude of CDDP-induced enhancement of gene expression at a given MOIwas determined by calculating the enhancement index:${{Enhancement}\quad {index}} = \frac{\beta \text{-}{gal}\quad {activity}\quad {of}\quad {CDDP}\text{-}{treated}\quad {cells}}{\beta \text{-}{gal}\quad {activity}\quad {of}\quad {control}\quad {cells}}$

In Vivo CDDP-Induced Enhancement of β-gal Gene Expression

Subcutaneous (SC) tumor nodules were created by injecting 1×10⁷ H1299cells suspended in 100 μl of PBS into the dorsal flank SC tissue of nudemice (nu/nu, Charles River, Wilmington, Mass.) that had received 350Rads of total body irradiation. Tumors, approximately 250 mm³ in size,consistently formed within 3 to 4 weeks of tumor cell implantation.Intratumoral injections of Ad/CMV/β-gal (5×10⁸ viral particles) weredone at 0, 2, 4, and 6 days following an intraperitoneal injection ofCDDP (5 μg/g body weight in 100 μl of PBS) or PBS only (n=4 per groups).The tumors were excised 48 hours after adenovirus injection, washed incold PBS, embedded in tissue freezing medium (Fisher Scientific,Houston, Tex.) and snap-frozen by immersion in 2-methylbutane which waschilled over liquid nitrogen. Serial frozen sections (8 μl, thick) ofthe tumor mass obtained at 2-mm intervals were fixed and stained withX-gal as previously described (Ponder et al., 1991) and thencounterstained with nuclear fast red (PolyScientific, Bay Shore, N.Y.).Quantitative assessment of β-gal gene expression (blue cells) wasdetermined by digital image analysis (Samba 4000, Immuno Software,Version 3.0; Image Products International, Inc., Chantilly, Va.) foreach tumor section. In vivo β-gal gene transduction of tumor masses wascalculated as the mean of the percentage of blue cells per surface areaof representative sections. Tumor-bearing animals receivingintraperitoneal injection of PBS and subsequent intratumoral injectionof Ad/CMV/β-gal at identical time points, served as controls. Themagnitude of enhancement was calculated by dividing values from CDDP andAd/CMV/β-gal treated tumors by values from Ad/CMV/β-gal control treatedtumors.

Statistical Analysis

The results are presented as means±standard deviations. Analysis ofvariance (ANOVA) and Student's t-test were used for statisticalanalysis; p<0.05 is considered significant.

RESULTS

Time Course and Dose Response Analysis

The initial response of incubating the H1299 and H460 cells with CDDP at1 and 4 μg/ml for 24 hours, ranged from a significant depression ofgrowth to complete inhibition of proliferation, however, those cellswere viable as demonstrated by trypan blue exclusion. At CDDPconcentrations lower than 0.25 μg/ml, cell growth remained unchanged ascompared to control cells populations. A brief 24-hour exposure of H1299and H460 cells to CDDP (0.062 μg/ml) resulted in an increase of geneexpression in target cells infected by Adv/CMV/β-gal as quantitated byX-gal staining (FIG. 1A). Maximal enhancement (2 to 2.5 fold increase inthe percentage of positively stained cells as compared to concurrentcontrols) was noted when cells were infected with Adv/CMV/β-gal 48 hoursafter removal of CDDP (p<0.001). The enhancement effect was short-livedas the percentage of positively stained, treated cells, infected on day4 or 5 after CDDP exposure was similar to that of the control. There wasalso a clear CDDP dose-related enhancement of β-gal gene expression inH1299 and H460 cells when infected with Adv/CMV/β-gal 48 hours afterCDDP removal (FIG. 1B). Maximal enhancement occurred at a CDDPconcentration of 0.062 μg/ml, however, there was also an enhancement at0.0039 and 1 μg/ml. Optimal conditions for maximal enhancement of geneexpression thus appears to be at the CDDP concentration of 0.062 μg/mland gene transfer at about 48 hours after drug treatment.

Mechanism of Enhanced Gene Expression

To determine if prior incubation of target cells with CDDP would resultin an increase in the ability of cells to take up gene-deliveringvectors or an increase in the expression of successfully deliveredgenes, β-gal gene expression was quantitated in H1299 and H460 celllines treated with CDDP and then infected with Ad/CMV/β-gal atincreasing MOI's. If CDDP mediates an enhancement of adenovirus uptakeas compared to non-treated controls, then there should be a proportionalincrease in enhancement indices with increasing MOI's. On the otherhand, if prior CDDP exposure results in enhanced gene expression only,then the enhancement index should be independent of the increasing MOI.The analysis showed that the β-gal activity in H1299 infected cellsincreased as the MOI's were elevated from 5 to 100 (control H1299 cellsof 0.82±0.11 to 16.8±3.2 U/mg protein and CDDP-treated H1299 cells of2.2±0.40 to 42.0±2.6 U/mg protein), indicating unchanged enhancementindices (3 to 2.5) with increasing MOI's (FIG. 2A). Similarly, β-galgene expression in CDDP-treated H460 cells increased from 0.28±0.06 to12.2±2.1 U/mg protein, as compared to β-gal gene expression in controlH460 cells of 0.056±0.016 to 7.6±1.8 U/mg protein. The enhancementindices in H460 cells, therefore, decreased from a value of 5 at an MOIof 5, to a value of 1.6 at an MOI of 100 (FIG. 2B). This observationimplies that CDDP induces an increase in the expression of successfullydelivered genes and not an increase in adenovirus uptake.

Enhanced β-gal Gene Expression by DNA-Damaging Agents

Infection of H1299 cells that were similarly treated with otherantineoplastic agents such as vincristine, methotrexate, 5-fluorouracilor trnsplatinum with Ad/CMV/β-gal did not result in enhanced geneexpression over a wide range of drug concentrations (FIG. 3).Methotrexate and 5-FU are anti-metabolites which interfere withsynthesis of nucleic acid precursors; vincristine binds to and inhibitsmicrotubular formation and impairs cellular mitosis (Fritsch et al.,1993). On the other hand, exposure of H1299 cells to the DNA-damagingagents VP-16 (etoposide) and ionizing irradiation (at 4 and 8 Grey)enhanced gene expression in tumor cells infected with Adv/CMV/β-gal(FIG. 3). These pharmacological agents induce DNA strand breaks bydifferent mechanisms: CDDP causes intra- and inter-strand crosslinkingof DNA by forming guanine adducts and eventually DNA strand breaks(Eastman, 1990); Etoposide forms a tertiary complex between DNA andtopoisomerase II and eventually produces protein-linked DNA single anddouble strand breaks (Kaufman, 1989); ionizing irradiation stimulatesproduction of oxygen free radicals which react with macromolecules andinduces DNA damage (Cole et al., 1980).

Enhanced β-gal Gene Expression in Other Cell Types and With DifferentVectors

When CDDP-treated H1299 cells were incubated with 2 other gene deliverysystems (the conjugated Ad/PLL/DNA complex or the cationic phospholipidlipofectamine), β-gal gene expressing cells occurred at the same CDDPdoses and to the same magnitude as observed followingadenovirus-mediated gene transfer (FIG. 4A). This indicates that theenhancement by CDDP is not vector dependent. This CDDP-inducedenhancement of transgene expression following Ad/CMV/β-gal infection wasalso observed in other malignant cell lines tested under the sameexperimental conditions (FIG. 4B). The magnitude of maximal enhancementwas dependent on the cell line tested (ranging from 1.6-fold (A549) to2.7-fold (H1299)), but it consistently occurred when gene transfer wasperformed 48 hours after exposure to 0.062 μg/ml of CDDP. Similarexposure of stationary primary human bronchial epithelial cells (90%confluent in culture) to a low concentration (0.062 μg/ml) of CDDPfailed to enhance gene expression in these normal cells afterAd/CMV/β-gal infection (FIG. 4B). However, at higher drug concentrations(1 and 4 μg/ml), there was a dose-dependent elevation of the percentageof β-gal-positive cells that was 1.5- to 2.5-fold higher than thatobserved in unexposed NHBE cells.

In Vivo Enhancement of β-gal Gene Expression

To analyze the effect of CDDP on enhancing gene expression in tumorcells in vivo, a time course analysis was done. The time course ofenhanced β-gal expression in H1299 cells in vivo was similar to the invitro time course (FIG. 5). The overall gene expression in subcutaneousH1299 tumors of animals injected with PBS and Ad/CMV/β-gal slightlydecreased (29.2±9.0% on day 0 to 18.2±5.6% on day 6) over time as tumorscontinued to grow while the viral titer remained unchanged. There was nochange in the β-gal gene expression in H1299 cells when CDDP andAd/CMV/β-gal were concurrently administered (day 0). However, thepercentages of positively stained cells in tumors injected withAd/CMV/β-gal 2 and 4 days after CDDP administration were 1.6- to2.8-fold higher than the values of the respective controls (46.0±4.3 vs28.7±6.2 on day 2 and 70.2±11 vs. 25.0±7.0 on day 4, p<0.01). When genetransfer was performed 6 days after CDDP administration, the increase inexpression was slightly higher than that of the control tumors (23.4±5vs. 182.0±5.6, p (FIG. 5).

EXAMPLE 2: GENE THERAPY STRATEGY FOR HUMAN NON-SMALL CELL LUNG CANCER:COMBINATION OF SEQUENTIAL CISPLATIN ADMINISTRATION ANDADENOVIRUS-MEDIATED p53 GENE TRANSFER Materials and Methods

Cells and Culture Conditions

The human NSCLC cell line H1299 with a homozygous deletion of p53 genewas grown in RPMI-1640 medium supplemented with 5% heat-inactivatedfetal calf serum (FCS), 10 mM glutamine, 100 units/ml of penicillin, 100μg/ml of streptomycin, 0.25 μg/ml of amphotericin B (Gibco-BRL, GrandIsland, N.Y.) (RPMI-complete). The H322 cell line was maintained inRPMI-complete medium supplemented with 10% FCS. The 293 cells weremaintained in complete high glucose Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% heat-inactivated FCS.

Recombinant Adenovirus Production

The properties of the adenovirus Ad/CMV/p53 have previously beendemonstrated in the inventors' laboratory (Diller et al., 1990). Thereplication-defective E1A-deleted adenovirus d1312 was used as acontrol. The virus was amplified on a large scale on 293 cells accordingto methods previously reported (Zhang et al., 1995a). Viral titer wasdetermined by UV-spectrophotometric analysis (viral particles/ml). Theabsence of replicative competence adenovirus was verified by a PCR™technique previously reported by Zhang et al. (1995b). The purifiedvirus was stored in 10% glycerol at −80° C. Infection of H1299 cells wascarried out by dilution of the viral stock to appropriateconcentrations, followed by the addition of viral solutions to cellmonolayers incubated in 1 ml of RPMI-1640 medium containing 2% FCS. Thecells were incubated for 2 hrs at 37° C. in a 5% CO₂ incubator, afterwhich 2 ml of RPMI-1640 complete medium supplemented with 5% FCS wasadded into the wells.

Cell Proliferation Assay

H1299 cells were exposed to CDDP (0.062 μg/ml of medium) for 24 hrs.Cells were then washed twice with PBS, trypsinized and seeded in 6-wellplates (10⁵ cells/well). Forty eight hours later, cells were infectedwith Ad/CMV/p53 at the multiplicity of infection (MOI) of 5. Daily cellcounts were performed for 5 days following transfection to study thetumor suppression effect of the combination of sequential CDDP andadenovirus-mediated p53 gene transfer. Controls consisted of untreatedcells or cells exposed to CDDP only, cells transfected with Ad/CMV/p53only and cells transfected with d1312 (similar MOI) with or withoutprior CDDP treatment.

Western Blot Analysis

H1299 cells were infected with Ad/CMV/p53 (MOI=1) 48 hrs after CDDPexposure. Cells were then harvested at 6, 12, 24, 36 and 48 hrs aftertransfection for Western blot analysis of p53 protein expression, asdescribed elsewhere (Diller et al., 1990). Unexposed cells similarlytransfected with Ad/CMV/p53 were used as controls. The human NSCLC cellline H322 which overexpresses mutant p53 protein was used as a positivecontrol. Relative quantities of exogenous p53 protein were determined bydensitometer (Molecular Dynamics, Sunnyvale, Calif.).

p53 Immunocytochemical Staining

The infected cells were fixed 3.8% formalin at 12 and 24 hrs aftertransfection with Adv/CMV/p53 (MOI=5) with or without prior CDDPexposure and treated with 3% H₂O₂ in methanol for 5 minutes. TheVectastain kit (Vector Laboratories, Burlingana, Calif.) was used forthe immunocytochemical staining. The primary antibody used was the mousemonoclonal anti-p53 antibody Pab 1801 (Oncogene Science, Manhasset,N.Y.). The secondary antibody was the biotinylated anti-mouse IgGantibody (Vector Laboratories). The avidin-biotin-peroxidase complex(ABC) kit was used to detect the antigen-antibody complex. The cellswere counterstained with Harris Hematoxylin (Sigma, St. Louis, Mo.) andmounted with cover slips using Cytoseal 60.

In Situ TUNEL Assay for Apoptosis

CDDP-treated or control H1299 cells were fixed in 50% acetone/ethanolfor 20 minutes at −20° C. 12 hrs and 24 hrs after transfection withAdv/CMV/p53 (MOI=5). In situ TdT (terminal deoxynucleotidyltransferase)-mediated dUTP-biotin nick end-labeling (TUNEL) assay wasperformed according to the procedure described elsewhere (Gavrieli etal., 1992). H1299 cells used as positive controls were treated withDNAse I (Gibco BRL, Gaithersburg, Md.), for 1 hr at 37° C. (50 μg/ml in10 mM Tris-HCI pH=7.5; 140 mM sodium cacodylate, 4 mM MgCl₂, 10 mMdithiothreitol).

In Vivo Combination of Sequential CDDP and Adv/CMV/p53 Administrations

H1299 tumor xenografts were created by injecting 1×10⁷ cells suspendedin 100 μl of PBS into the dorsal flank subcutaneous space of nude micethat had received 350 Rads of total body irradiation prior to injection.Subcutaneous tumor nodules of 200 to 250 mm³ in size were formed 3 to 4weeks later. Different strategies of CDDP and Ad/CMV/p53 combinationwere studied for their tumoricidal efficacy: a) intraperitoneal (ip)CDDP (5 μg/g body weight) given on day 0 and followed by intratumoralAd/CMV/p53 injections of 1.5×10¹⁰ viral particles/100 μl PBS in a singledose on day 3 or in 3 equally divided doses of 5×10⁹ viral particles/100μl PBS on days 2, 4 and 6; b) simultaneous CDDP and Ad/CMV/p53administrations in single or 3 equally divided doses (divided CDDPdoses: 1.67 μg/g body weight) and c) ip CDDP given 3 days aftercompletion of intratumoral Ad/CMV/p53 injections. The divided doseregime was designed to address issues that may limit the use of highvirus titer and volume of injectate such as toxicity and the low titerof viral stock. The controls consisted of tumors injected with eitherPBS, Ad/CMV/p53 without prior systemic CDDP, d1312 with or without priorip CDDP or tumor-bearing animals receiving ip CDDP only. Tumor sizeswere measured every 2 days for 32 days and tumor volumes were estimatedby assuming a spherical shape with the average tumor diameter calculatedas the square root of the product of the orthogonal diameters. Allanimals were treated according to guidelines developed by the M. D.Anderson Animal Care and Use Committee. All mice were sacrificed whentumors grew to 4000 mm³.

Statistical Analysis

Results were presented as mean±standard deviation. Analysis of variance(ANOVA) and two-tailed Student's t test were used for statisticalanalysis of multiple groups and pair-wise comparison respectively,p<0.05 is considered significant.

RESULTS

In Vitro Proliferation Assay

H1299 cells were treated with a combination of sequential CDDP andAd/CMV/p53 infection and then analyzed by cell proliferation assay todetermine if the combination of CDDP and adenovirus-mediated p53 genetransfer would result in a superior growth-inhibiting effect. TreatingH1299 cells with CDDP (0.0625 μg/ml) for 24 hrs had no effect on cellgrowth in vitro, nor did mock infection with d1312 with or without priorCDDP treatment. On the other hand, exposure of H1299 cells to CDDP 48hrs prior to Adv/CMV/p53 transfection resulted in 61% and 55% increasedinhibition of tumor proliferation on day 3 and 5 after p53 transfer whencompared to control H1299 cells similarly transfected with Adv/CMV/p53(FIG. 6).

Western Blot Analysis and p53 Immunocytochemical Staining

The effect of CDDP treatment on levels of p53 protein was determined inCDDP-treated and untreated control cells by Western blotting of celllysates harvested at different time points after Adv/CMV/p53 infection.Expression of the p53 gene occurred as early as 6 hours afterAdv/CMV/p53 transfection in both CDDP-treated and control H299 cells.However, prior exposure to CDDP led to a higher level of p53 at 12, 24,36 and 48 hrs after transfection. Densitometry analysis showed that therelative levels of p53 protein (normalized for β-actin levels) inCDDP-treated cells were 0.73, 1.39, 1.49. 1.20 compared to 0.23, 0.90,0.68, 0.62 (for a 2- to 3-fold increase in the levels of p53 protein) incells without prior CDDP at each of the time points studied. The p53levels at 6 hrs after transfection were too low to be analyzed. CDDPtreatment appeared to significantly increase the p53 gene expression butdid not seem to alter the transduction kinetics of H1299 cells.Immunocytochemical staining of H1299 cells 24 hrs after p53 genetransfer demonstrated that almost all of the CDDP-treated cells stainedpositive, at a significantly higher intensity, for the p53 protein ascompared to cells not exposed to CDDP. This elevation of p53 geneexpression correlates well with the enhanced tumoricidal effect of thecombination of CDDP and Adv/CMV/p53 in vitro as demonstrated in FIG. 6.

Induction of Apoptosis Following Adv/CMV/p53 Transfection

The in situ TUNEL assay was used to analyze the extent of apoptosis thatoccurred in Ad/CMV/p53-transfected H1299 cells. In cells that were notexposed to CDDP prior to gene transfer, apoptosis began 12 hrs after p53transfection with few TUNEL-positive cells being visualized withsignificant apoptosis was detected 24 hrs after Ad/CMV/p53 transfection.Apoptotic cells could be readily seen in CDDP-treated cells as early as12 hrs after transfection, at a much higher frequency than in untreatedcells. By 24 hrs, almost all CDDP-treated cells stained positive for DNAfragmentation. The degree of apoptosis and the timing of its occurrencefollow very closely the time course of p53 gene expression inCDDP-treated cells. Thus enhanced and accelerated p53 gene expression incells treated with sequential CDDP and Ad/CMV/p53 most probably resultedin an early and intense induction of apoptosis which translated to anincreased tumoricidal effect.

Inhibition of Tumor Growth In Vivo

Divided-Dose Regime: Tumors grew rapidly in groups of control animalsreceiving only ip CDDP or intratumoral PBS with tumor sizes reaching themaximal allowable volume of 4000 mm³22 to 26 days after treatment.Tumors injected with d1312 with or without prior ip CDDP showed somedegree of growth retardation (up to 30% of normal tumor growth)secondary to vector toxicity. Injections of Ad/CMV/p53 (1.5×10¹⁰ viralparticles in 3 equally divided doses) resulted in inhibition of tumorgrowth during and immediately after the treatment. These tumors,however, resumed growth at the normal rate, 10 days after the lastinjection; reaching the mean tumor volume of 3357±391 mm³32 days afterthe onset of therapy. Intraperitoneal CDDP administration given 2 daysprior to the beginning of the gene therapy schedule resulted in a morepronounced inhibition of tumor development. There was a regression oftumor mass that lasted for 14 days before the tumor growth resumed whichwas at a much slower rate. At the end of the observation period, theaverage tumor size was 1497±221 mm³ (p<0.001vs Ad/CMV/p53 without priorCDDP). Systemic CDDP administration prior to p53 gene replacementtherapy, therefore, resulted in a synergistic effect of tumor growthinhibition that was responsible for at least a 55.4% further reductionin tumor size.

Single-Dose Regime: When Ad/CMV/p53 was given in a single dose,inhibition of tumor growth was greater than that seen with thedivided-dose regime: 1936±308 vs 3357±391 (without CDDP) and 773±197 vs1497±221 (with prior CDDP) respectively. The combination of CDDPadministration 3 days prior to a single intratumoral injection ofAd/CMV/p53 (1.5×10¹⁰ viral particles) resulted in a 60% furthersuppression of tumor growth compared to tumors treated with Ad/CMV/p53without prior CDDP. To illustrate the importance of the timing of CDDPadministration in relation to p53 gene transfer, CDDP (5 μg/ml) wasgiven at the same time as Ad/CMV/p53 (1.5×10¹⁰ viral particles)injections in a second group of mice, and in a third group CDDP wasgiven 3 days after adenovirus-mediated p53 gene transfer. Thecombination of sequential CDDP and Ad/CMV/p53 showed the mostsignificant tumor growth inhibition effect with the mean tumor volume of773±197 mm³, compared to 1257±225 mm³, when CCDP was given concurrentlywith adenovirus injections, and 1750±214 mm³, when CDDP was given afteradenovirus-mediated p53 transfer (p<0.001 by ANOVA). Ad/CMV/p53significantly prolonged the survival of treated animals to 62.8±4.6 days(range: 60 to 70 days) after the onset of therapy compared totumor-bearing mice injected with Ad/CMV/p53 alone of 46.0±4.4 days oruntreated controls of 23.4±2.9 days (p<0.001).

H. References

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What is claimed is:
 1. A method for enhancing the expression of atransgene in a target neoplastic cell in vivo comprising: (a)administering a DNA-damaging agent to a subject containing a targetneoplastic cell; and (b) transferring said transgene into said targetneoplastic cell between 2-4 days after said administering step; wherebyexpression of said transgene is enhanced as a result of theadministering of said DNA-damaging agent to said target neoplastic cell.2. The method of claim 1, wherein said target neoplastic cell is adividing cell.
 3. The method of claim 1, wherein said DNA-damaging agentis selected from the group consisting of cisplatin, carboplatin, VP16,teniposide, daunorubicin, doxorubicin, dactinomycin, mitomycin,plicamycin, bleomycin, procarbazine, nitrosourea, cyclophosphamide,bisulfan, melphalan, chlorambucil, ifosfamide, merchlorehtamine, andionizing radiation.
 4. The method of claim 1, wherein said transgene istransferred at about 3 days after said administering step.
 5. The methodof claim 1, wherein said transfer of said transgene is accomplished by atechnique selected from the group consisting of liposome-mediatedtransfection, receptormediated internalization and viral infection. 6.The method of claim 1, wherein said transgene encodes a tumorsuppressor.
 7. The method of claim 6, wherein said tumor suppressor isp53.
 8. The method of claim 7, wherein said p53 transgene is under thetranscriptional control of a CMV IE promoter.
 9. The method of claim 3,wherein said DNA-damaging agent is cisplatin.
 10. The method of claim 7,wherein said p53 transgene is carried in an adenoviral vector.